1. Field of the Invention
The present invention generally relates to anti-tumor peptides. In particular, the present invention relates to use of specified pro-IGF-1-peptides in the treatment and amelioration of tumor-producing diseases. Certain pro-IGF-1-peptides are shown to decrease tumor growth and to reduce metastasis.
2. Brief Description of the Related Art
Attention recently has been focused on the biological activities of the proteolytically-processed polypeptides from post-translational modified peptide hormones. For example, the C-peptide of pro-insulin has long been regarded to be biologically inactive except for a possible role in the folding of the insulin molecule during its post-translational modification. However, Ito et al. (14) have found recently that the C-peptide of pro-insulin is important in restoring vascular and neural dysfunction and Na+/K+-dependent ATPase activity in diabetic rats. Although a synthetic peptide amide of human b-type IGF-I E-peptide has been shown to exert mitogenic activity by Sieffied et al. (5), the biological activity of the native human E-peptides have not been identified.
Insulin-like growth factor-I (IGF-I) is a member of the insulin gene family and plays an essential role in growth, differentiation, regeneration and metabolism in all vertebrates (2). In all animals studied to-date, the primary translation product of IGF-I mRNA contains distinct regions or domains, including an N-terminal pre-peptide leader, followed by the mature peptide with B, C, A and D domains, and a C-terminus E domain. Due to alternative RNA splicing, two different forms, the a- and b-type, of IGF-I pro-hormones exist in mammals, differing only in the E-peptide region. In human, mouse and rat, the first 15 amino acid residues in the N-termini of the E-peptides of pro-IGF-Ia and -Ib share identical sequences, and this region is referred to as the common region. The sub-types of pro-IGF-I""s are distinguished from one another by the amino acid sequences after the first 15 common amino acids, known as the variable regions of the E-peptides (3).
In mammals, the signal peptide and the E-peptide are proteolytically cleaved after translation from the pre-pro-hormone to form a mature peptide of 70 amino acid residues. The E-peptides of mammalian pro-insulin-like growth factor-I (pro-IGF-I) have long been regarded as biologically inactive.
Surprisingly, the present inventors have recently discovered that certain recombinant rainbow trout pro-IGF-I E-peptides (Ea-2-, Ea-3- and Ea-4), like hIGF-1, exhibit a dose-related mitogenic activity in several mammalian cell lines (Tian et al. (1)).
Four different forms of a-type IGF-I mRNA have been observed in rainbow trout (rt), Oncorhynchus mykiss, and are designated as IGF-I Ea-1, Ea-2, Ea-3 and Ea-4 (4). Nucleotide sequence comparison of the four size forms of IGF-I mRNAs reveal that the size differences among these mRNA species are due to insertions or deletions in the E domain regions of the molecules. The first 15 predicted amino acid residues of the four E-peptides share identical sequences among themselves as well as with pro-IGF-I E-peptides of human, mouse, and rat. The presence of the C-terminal 20 amino acid residues, sharing 70% identity with their human counterparts, identifies them to the a-type E-peptides.
The Ea-1-peptide of the rt-pro-IGF-I is a polypeptide of 35 amino acid residues, comprising the first 15 and the last 20 amino acid residues. Ea-2 and Ea-3 peptides differ from Ea-1 by virtue of either a 12- or 27-amino acid residue insertion between the first and last segments of the Ea-1-peptide sequence, respectively, while Ea-4 contains both insertions. The predicted numbers of amino acid residues in each E-peptide are, thus, 35, 47, 62 and 74, respectively. There has not been any report on the presence of b-type IGF-I mRNA in rainbow trout.
While the biological functions of the mature IGF-I peptide have been intensively studied, the functions of E-peptides have been overlooked and remain to be identified.
Siegfried et al. (5) have recently shown that the amide of a 22-amino acid residue encoded within the variable region of the E-peptide of the human IGF-Ib (IBE1) exerts mitogenic activity in normal and malignant bronchial epithelial cells. Surprisingly, as set forth above, the present inventors have recently found that certain E-peptides from fish possess mitogenic activity with respect to mammalian cells (Tian et al. (1) report that recombinant rainbow trout Ea-2-, Ea-3- and Ea-4-peptide possess mitogenic activity in several normal and oncogenic transformed mammalian cell lines including NIH 3T3 cells, human embryonic kidney cells transformed by retrovirus (293GP), human mammary gland tumor cells (MCF-7) and caprine mammary epithelium cells (CMEC) (6)).
Based on the surprising inter-class activity of icthyofauna Pro-IGF-I E-peptides on mammalian cells, and given that trout Ea-2, Ea-3 and Ea-4 peptides containing a signal motif for peptidyl C-terminal amidation (4,7) and a bipartite consensus nuclear localization sequence (4,8), the present inventors hypothesized that these peptides might possess other novel biological activities on mammalian cells. In its screening for novel biological activities of trout pro-IGF-I E-peptides, the present inventors undertook several studies of the effects of such E-peptides on morphology, colony formation activity on a soft agar medium, and invasiveness of human and trout cancer cells. Surprising and unexpected anti-tumor activity was discovered.
The present provides a method for reducing tumor growth and the invasiveness of cancerous cells in mammals and fish by administration of a Pro-IGF-I E-peptide obtained from fish.
The present inventors have shown that the treatment of established human and fish cancer cell lines (MCF-7; HT-29; HepG2, ZR-75-1, SK-N-F1 and HC) and an oncogenic transformed human cell line (293GP) with recombinant trout Ea-2- or Ea-4-, but not Ea-3-peptide, resulted in a dose-dependent induction of morphological change and enhanced cell attachment. They have also shown that the in vitro colony formation activity of the oncogenic transformed cell line or established tumor cell lines is greatly reduced or diminished by treatment with the trout Ea-4-peptide. Furthermore, the invasive activity of HT1080, a known invasive cancer cell line, is also reduced many fold by treatment with the Ea-4-peptide. These results suggest that the Ea-4- and Ea-2-peptides of rainbow trout pro-IGF-I are able to control the malignant properties of cancer cells. The E-peptide-induced morphological changes are sensitive to treatment with xcex1-amanitin or cycloheximide, known inhibitors of RNA and protein synthesis.
In one embodiment of the present invention there is disclosed a method of inhibiting proliferation of malignant cells, comprising administering to the malignant cell at least one E-domain peptide agent. The E-domain peptide agent is preferably selected from the group consisting of: a trout E-domain peptide, an E-domain peptide homolog, and an E-domain peptide fusion protein, and may more preferably be an E-domain peptide of a rainbow trout. In a preferred embodiment, trout E-domain peptide is selected from the group consisting of: Ea-2 domain peptide (SEQ ID NO:2) and Ea-4 domain peptide (SEQ ID NO:4). The E-domain peptide agent may be administered in a pharmaceutical composition.
In another embodiment of the present invention, there is disclosed method of inhibiting the proliferation of malignant cells, comprising administering to the malignant cells a nucleic acid encoding an E-domain peptide agent. Preferably the E-domain peptide agent is selected from the group consisting of: a trout E-domain peptide, an E-domain peptide homolog, and an E-domain peptide fusion protein.and more preferably the E-domain peptide is an E-domain peptide of a rainbow trout, in particular having a sequence selected from the group consisting of: Ea-2 domain peptide (SEQ ID NO:2) and Ea-4 domain peptide (SEQ ID NO:4). The nucleic acid encoding the E-domain peptide may be administered in a pharmaceutical composition.
In yet another embodiment of the present invention, there is disclosed a method for reducing the invasiveness of malignant cells, comprising administering to the malignant cells an E-domain peptide. In such embodiment, the E-domain peptide agent is preferably selected from the group consisting of: a trout E-domain peptide, an E-domain peptide homolog, and an E-domain peptide fusion protein, and more preferably is an E-domain peptide of a rainbow trout. Preferably, the trout E-domain peptide is selected from the group consisting of: Ea-2 domain peptide (SEQ ID NO:2) and Ea-4 domain peptide (SEQ ID NO:4). The E-domain peptide agent may be administered in a pharmaceutical composition.
And yet in another embodiment of the present invention, there is disclosed a method for reducing the invasiveness of malignant cells, comprising administering to the malignant cells a nucleic acid encoding an E-domain peptide agent. Preferably, the E-domain peptide agent is selected from the group consisting of: a trout E-domain peptide, an E-domain peptide homolog, and an E-domain peptide fusion protein, and more preferably is an E-domain peptide of a rainbow trout. Preferably, the trout E-domain peptide is selected from the group consisting of: Ea-2 domain peptide (SEQ ID NO:2), and Ea-4 domain peptide (SEQ ID NO:4). The nucleic acid encoding the E-domain peptide may be administered in a pharmaceutical composition.